![]() In order to explore the interaction of human HIRA with its known co-subunits, we generated two YFP-tagged HIRA constructs corresponding to the amino acids 1–440 and 492–1017 that contain the interaction domains for UBN1 and CABIN1, respectively 15, 19 (Fig. The HIRA subunit homooligomerizes in cells We thus provide a quaternary structure of the HIRA subunit with major implications for the functional activity of the complex. This homotrimerization is critical for CABIN1 interaction, which binds with a 3:2 HIRA–CABIN1 stoichiometry in vitro and key for HIRA enrichment at UV damage sites and for efficient new H3.3 deposition. By using biochemical and structural approaches, our study reveals that the HIRA subunit forms a homotrimer with homology to the replisome component Cohesion establishment factor 4/Acidic nucleoplasmic DNA-binding protein-1 (Ctf4/AND-1). Finally, the H3.3 deposition activity of the complex has been reported to be modulated by post-translational modifications of the HIRA subunit 25, 26.Ĭonsidering the central position of HIRA, and its multiple partners, a major question to address remained despite these studies, namely to decipher the functional oligomerization state of the HIRA complex. ![]() The fact that the HIRA complex associates with various proteins, such as transcription factors, RNA pol II, Prohibitin (PHB), and replication protein A (RPA) suggests that the targeting of the complex at specific locations may exploit additional structural properties and mechanisms 5, 21, 22, 23, 24. The DNA-binding capacity of the complex has been proposed to promote a general recruitment at any transient nucleosome-free region for H3.3 deposition 5, 20. ![]() Depletion of HIRA leads to the co-depletion of both CABIN1 and UBN1 proteins further underlining the fact that HIRA plays a central role 5. It binds UBN1/2, CABIN1, and ASF1a through its N-terminal WD40 repeat, C-terminal C, and central B domains, respectively 8, 15, 19. Furthermore, HIRA serves as a scaffold protein to bring together the other subunits as well as ASF1a. However, its specific function in the HIRA complex remains to be determined. ![]() CABIN1, originally identified as a Calcineurin-binding protein whose interaction is dependent on protein kinase C (pKC) and calcium signals, negatively regulates the activity of other interactors, such as Myocyte enhancer factor 2 (MEF2) and p53 16, 17, 18. However, whether UBN1 and UBN2 are present together in the same complex or constitute distinct entities is not known. Notably, in addition to UBN1, another Ubinuclein exists, UBN2, that is also able to interact with HIRA 15. Within the HIRA complex, a Hpc2-related domain (HRD) of UBN1 interacts directly with H3.3 as revealed by in vitro biochemical studies and X-ray crystallography 14. Moreover, it has been involved in either the activation or the long-term maintenance of gene expression patterns 11, 12 and HIRA depletion has been reported to affect transcription recovery after DNA repair 13. The HIRA-mediated H3.3 deposition has been strongly associated with actively transcribed genes 10. ASF1a interacts specifically with the HIRA subunit connecting this chaperone with the DNA synthesis-independent assembly pathway mediated by the HIRA complex 8, 9. In addition to the HIRA complex, other histone chaperones, in particular Anti-silencing factor 1 (ASF1a and ASF1b), have been purified with H3.3 3. Notably, these three components have counterparts in yeast that constitute the HIR complex 7. HIRA is in a complex that contains at least two other subunits, Calcineurin-binding protein 1 (CABIN1) and Ubinuclein 1 (UBN1) 6. HIRA promotes histone deposition independently of DNA synthesis as shown in Xenopus laevis egg extracts 4 and deposits new histones H3.3 in both replicating and non-replicating human cells 5. The presence of the Histone regulator A (HIRA) with the histone variant H3.3 in a soluble complex has implicated this histone chaperone in the specific deposition of this variant 3. Histone chaperones are currently considered the most likely candidates responsible for histone variant deposition 1, 2.
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